Identification of 1 alpha,25-dihydroxyvitamin D-3 response elements in thehuman transforming growth factor beta 2 gene

Transforming growth factor-beta (TGF-beta) is one of the most abundant grow th factors secreted by bone cells, and regulation of TGF-beta expression is crucial for bone development and growth. Previous studies from our laborat ory demonstrated that 1 alpha,25-dihydroxyvitamin D-3 (1 alpha,25(OH)(2)D-3 ) inhibits human osteoblast and keratinocyte growth by increasing TGF-beta 2 secretion and synthesis. To examine the mechanism by which 1 alpha,25(OH) (2)D-3 regulates TGF-beta 2 transcription in osteoblasts, we ligated segmen ts of the human TGF-beta 2 promoter 5' of a growth hormone reporter gene in a growth hormone reporter plasmid and examined the effects of 1 alpha,25(O H)(2)D-3 administration on the expression of growth hormone in cells transf ected with such chimeric promoter-reporter plasmids. The promoter region ex tending from -973 to +68 bp of the transcription start site elicited a 7-fo ld increase in reporter gene activity in transiently transfected osteoblast s after 1 alpha,25(OH)(2)D-3 treatment, whereas the region from -553 to f68 bp of the transcription start site showed no response following 1 alpha,25 (OH)(2)D-3 treatment. Transfection of osteoblasts with reporter plasmids co ntaining TGF-beta 2 promoter DNA from -869 to -658 bp revealed a 3.8-fold i ncrease in reporter gene activity. DNA fragments from this region (-743 to -676 bp and -786 to -728 bp) ligated into reporter plasmids also showed inc reases in reporter activity. Gel retardation assay experiments showed that DNA fragments from -774 to -735 bp and -714 to -675 bp both formed a DNA-pr otein complex with bacterially expressed human retinoic acid X receptor alp ha (RXR alpha) and vitamin D receptor (VDR) and with nuclear extracts from human bone cells. Addition of a vitamin D receptor antibody to reactions co ntaining the aforesaid DNA fragments and bone cell nuclear extract resulted in further retardation of the mobility of the DNA-protein complex (super-s hifting). Removal of two putative direct repeat DNA fragments in this regio n abolished VDR-RXR alpha-vitamin D response element complex formation. The TGF-beta 2 promoter contains two imperfect direct repeat DNA sequences: <( TGTAGA)under bar>ACA<(AGTAGA)over bar> and <(AATGAA)over bar>GTT<(GGTGGA)ov er bar> that mediate the effect of 1 alpha,25(OH)(2)D-3.